PROJECT SUMMARY/ABSTRACT. Inflammation is our primary response from the innate immune system to fight infection and self-protect from damage. However, dysfunctional regulation of inflammation results in disease, including certain types of cancer, autoimmune, cardiovascular and neurological disorders, rheumatoid arthritis, and even depression. The onset of inflammation depends on the assembly of a multiprotein complex known as the inflammasome. The main players in inflammasome assembly are; - sensor proteins that react upon danger signals derived from pathogens or damaged tissue; - procaspase-1 that activates inflammatory cytokines as a result of inflammasome assembly; - the adapter ASC that functions like a molecular glue by connecting sensor and procaspase-1 molecules. Canonical ASC has an isoform, which shows different self-assembly capabilities and modulates the intensity of inflammation in the cellular context. The presence of protein isoforms is a well-known, natural mechanism for the regulation of protein function. Both proteins are bimodular with two Death Domains connected by a linker, and their amino acid sequences differ solely in the linker length. Canonical ASC has a 23 amino acid-long linker, whereas its isoform has a shorter linker of 3 amino acids (ASC_short). We demonstrated that ASC linker plays a key role in defining the orientation of its domains, and show in this proposal that both domains actively participate in ASC self-assembly to form filamentous macrostructures. Evidence indicate that inflammasomes assemble into different supramolecular structures. However, little is known on the factors controlling these structural arrangements, or how the different assemblies impact inflammation. This proposal aims at addressing a knowledge gap in the role of ASC isoforms on inflammasome structural organization and inflammatory response. The long-term goal of this proposal is to gain in-depth knowledge on the interplay between the inflammasome components to understand its function and regulation, which is of great significance to set the grounds for the development of therapeutics to control inflammation. The objective of this grant is innovative because it will decipher the unknown molecular bases for inflammasome regulation mediated by ASC isoforms. Our hypothesis is that; 1) the linker length has important implications in the interaction properties of ASC and ASC_short at the molecular level, which can account for the observed alteration of the inflammatory response, 2) the linker length leads to differences in the interdomain dynamics of the two isoforms and in the resulting macrostructures. To test this hypothesis we propose to; 1) determine the inflammatory activity, precise self-association and interacting capabilities of ASC isoforms, including ASC, ASC_short and an artificial form of ASC engineered with a long linker (3 times the canonical linker length: ASC_long) as a reference for independent domains, 2) determine the interdomain dynamics of the three isoforms using Nuclear Magnetic Resonance; 3) discern with Transmission Electron Microscopy the potentially different characteristics of the macrostructures resulting from ASC isoforms self-association, and their implication in inflammatory activity.